The invention is in the field of electrophoresis. It is of particular interest in terms of its application in genetic engineering and molecular biology.
Additional information pertinent to this invention may be found in Schwartz, D. C. and Cantor, C. R., "Separation of Yeast Chromosome-Sized DNAs by Pulsed Field Gradient Gel Electrophoresis," Cell, Volume 37, pg. 67, May 1984; and Van Der Pleog, H. T., Schwartz, D. C., Cantor, C. R. and Borst, P., "Antigenic Variation in Trypanosoma brucei Analyzed by Electrophoretic Separation of Chromosome-Sized DNA Molecules," Cell, Volume 37, pg. 77, May 1984.
The invention which is based upon the discovery of a new kind of electrophoresis makes it possible, inter alia, to carry out important analyses which were not possible or practical with previously known techniques. Potential applications include the separation of chromosomal DNA, chromosomal mapping, the convenient production of genetic libraries, studies on the effects of various drugs on chromosomal DNA, and the convenient characterization of polymers. The invention makes it possible to separate with a high degree of resolution and at high speeds larger particles (molecules) than those capable of resolution with prior art techniques and to concurrently separate particles which differ substantially in mass. In a preferred embodiment, the invention makes it possible to lyse cells for electrophoretic separation of macromolecules e.g. chromosomes contained within the cells with minimal degradation or breakage.
Electrophoresis in which particles such as a mixture of macromolecules are moved, e.g., through a gel matrix, by an electric field, is a widely used technique for qualitative analysis and for separation, recovery and purification. It is particularly important in the study of proteins, nucleic acids and chromosomes. See, e.g., Cantor, C. R. et al., Biophysical Chemistry, Freeman, 1980, Part 2, pp. 676, 683. Indeed, it is probably the principal tool used in most DNA and chromosomal analysis.
The particles to be analyzed and separated by electrophoresis are placed in a support medium such as a gel and are subjected to an electric potential. Difficulties arise when electrophoretic separation of very large particles is attempted. For example, using previously known techniques, the size of the largest DNA molecule routinely handled is that of a bacteriophage (3.2.times.10.sup.7 daltons). Such a limit on size prevents many kinds of desirable analyses from being carried out. For example, intact chromosomal DNAs are larger and are typically reduced in size in order to make it possible to work with them. This, however, destroys important information encoded within the DNA and precludes many important experiments and analyses.
Methods of extending gel electrophoresis to particles of higher mass by reducing the gel concentrations have been proposed. However, this adversely affects resolution, makes experimental conditions difficult to control and has not been successfully applied to DNA molecules having molecular weight greater than about 5.times.10.sup.8 daltons. Fangman, W. L., Nucleic Acids Research, Vol. 5, No. 3, March 1978, pp. 653-655; Serwer, P., et al., Electrophoresis, 1981, Walter, deGreuyter and Coe, pp. 237-243.
It is believed that resolution in previously known electrophoresis techniques is field-dependent since lower electric field intensities generally give higher resolution. As a consequence, electrophoresis runs in which higher resolution is desired often take as long as 100 hours. Moreover, particle mobility, and hence resolution capability, is believed to vary with the logarithm of the mass of the particles to be separated, which of course is not a highly sensitive basis for obtaining separations. Additionally, in known prior art gel electrophoresis, different gel concentrations are typically used for different mass or molecular weight ranges, thereby limiting the range of particles which can be concurrently resolved. Furthermore, previously known electrophoresis techniques are typically used to separate only small amounts of particles, and the process cannot conveniently be extended to larger amounts.
Another problem involved in the electrophoretic separation of large molecules e.g. DNA arises because the molecules (DNAs) must first be isolated since they may not exist as free molecules in the cell. For cells such as yeast and bacterial cells which have a cell wall isolation of DNA generally involves weakening the cell wall by treating it with an enzyme such as lysozyme for bacteria or zymolyase for yeast to form spheroplasts and with a chelating agent e.g. ethylenediaminetetraacetic acid (EDTA). For cells such as mammalian cells which do not have a well-defined cell wall it is of course not necessary to carry out such a treatment step. Cell lysis of spheroplasts or of cells which do not have a well-defined cell wall may be then accomplished by the addition of a detergent such as sodium dodecyl sulfate (SDS) in a buffered saline solution.
Following lysis, the solution is treated with pancreatic ribonuclease to hydrolyze RNA with protease to degrade proteins. Residual proteins and oligopeptides are extracted with an organic solvent, such as phenol or a mixture of equal volumes of phenol and chloroform. Most of the protein will denature and enter the organic phase or precipitate at the interface of the organic and aqueous phases. The clear, viscous aqueous phase containing the DNA may be removed. With the addition of alcohol, the DNA will precipitate out of the aqueous phase as a white fibrous material and may be spooled out on a glass rod. Precipitation from alcohol serves to concentrate the high molecular weight DNA while removing the small oligonucleotides of DNA and RNA, detergent and the organic solvent used in the removal of proteins. Residual detergent and salts may be removed by dialysis of the resuspended DNA solution against the desired buffer. In some instances, it may be desirable to further purify the DNA by centrifugation on isopycnic cesium chloride gradients or hydroxylapatite chromatography.
DNA molecules are extremely susceptible to breakage from shearing forces. As can be seen from the foregoing description of the conventional method for isolating DNA molecules, excessive amounts of shearing forces are applied to the DNA molecules because of the numerous manipulations involved. This results in considerable breakage of the DNA molecules.
Despite the fact that electrophoresis has been used for some time, and despite the fact that important limitations thereof and the need to overcome them have also been long known, no previous proposals are known which have successfully overcome such limitations.
In one embodiment, this invention is a significant departure from the established principles of electrophoresis and is based on the surprising discovery that electrophoresis through deliberately varied electric fields, rather than through the uniform fields sought in previously known electrophoresis methods, unexpectedly yields highly desirable results. More specifically, the invention is based on the discovery that desirable separation results when particles are subjected to respective electrical fields which move them in overall directions generally transverse to the respective general directions of the fields. Particularly desirable results are achieved in at least those cases examined to date when at least one of the electric fields has a deliberate intensity gradient in a direction transverse to its own. As a specific nonlimiting example, two fields can be used which alternate between respective high and low intensities out of phase with each other and are in directions transverse to each other. For example, one of the fields can be on while the other one is off, etc. Particularly good results are obtained when the on and off times of the fields are related to the mass of the particles to be separated, e.g., when the on and off periods are proportional to the mass of the particles raised to a power of about 1.5.
One of the important advantages of this discovery is that it dramatically extends the mass range of particles which can be electrophoretically separated at high resolution. As a nonlimiting example, the new technique can separate at high resolution particles whose mass is about 1.2.times.10.sup.9 daltons, while the upper limit of previously known methods which provide lower resolution, is believed to be about 0.5.times.10.sup.9 daltons. It is believed that the new technique can also resolve particles larger than 1.2.times.10.sup.9 daltons. Another important advantage is that in the new technique, resolution is much less dependent on electric field intensity; consequently, the new kind of electrophoresis can be run at much higher speed, so long as heat produced can be effectively dissipated. As a result, a typical laboratory run can be carried out in 4 to 8 hours, while corresponding runs using prior art techniques require 12 to 100 hours. Another significant advantage of the new technique is that larger amounts of sample, as compared to the known prior art, can be used, thus giving increased resolution and sensitivity. A further advantage is that the new technique can simultaneously resolve, in the same gel, particles from a wider mass range than is believed possible with prior art techniques. As a nonlimiting example, the new technique can resolve simultaneously, in the same gel, particles ranging in mass from about 10.sup.6 to about 10.sup.9 daltons. With previously known techniques several different gel concentrations would have been required to resolve particles in the narrower mass range from about 10.sup.6 to about 10.sup.8 daltons.
As yet another important aspect of the invention, a technique has been found to minimize handling damage to cell derived macromolecules such as DNAs by lysing cells or spheroplasts, in the case of cells having well-defined cell walls, which have been entrapped in a suitable matrix such as a block of gel which is the same as, or compatible with, the electrophoresis gel, and implanting the entire block in the electrophoresis chamber. Another important aspect of this invention is that the blocks of gel may be formed automatically and may be inserted into the electrophoresis chamber automatically with no significant damage to cell-derived macromolecules.
The advantages of releasing macromolecules such as DNA in situ in a solidified gel insert are considerable. For example, the DNA is rendered very stable and may be stored for weeks or even months at room temperature or for days or even a few weeks at temperatures as high as 50.degree. C. This provides major advantages for using electrophoretic methods such as the method of this invention in applications such as in diagnostic applications since DNA entrapped in solid gel inserts may be conveniently shipped from one location to another, thus permitting widespread sample collection and subsequent shipment of samples to a central location for analysis or for storage in a DNA bank where the samples may be maintained indefinitely at low temperatures without risk of damage to the DNA or cross contamination of samples which can easily occur when dealing with liquid samples. Moreover, such solid samples eliminate the need to accurately measure liquid samples and thus reduce error associated with variations in sample size.
The advantages of the use of the gel inserts of the present invention also extend to the ease with which electrophoretic methods may be automated to take advantage of stable and uniformly shaped, modular samples of routine analysis, thus creating a major opportunity for carrying out disease diagnosis at the molecular level by analysis of chromosomal DNAs. Specifically, the DNA in the inserts may be treated in situ with restriction enzymes to produce large intact fragments which can be used for detailed biochemical and molecular analysis, a result not possible with prior techniques. Moreover, the advantages of this approach extend although perhaps to a lesser extent to other macromolecules such as RNAs.
These and other advantages of the invention, as well as additional inventive features, will become apparent from the detailed description which follows.